In recent years, proteins with aberrant glycosylation patterns have emerged as therapeutic targets in oncology. Along these lines, MUC16 glycoforms have been implicated in the oncogenesis and metastatic progression of high-grade serous ovarian cancer (HGSOC) and pancreatic ductal adenocarcinoma (PDAC). AR9.6 is a therapeutic mAb that binds to MUC16, interferes with the interaction of MUC16 with ErbB receptors on the surface of cancer cells, and thereby attenuates the activation of downstream oncogenic signaling pathways. Here, we report the in vitro, ex vivo, and in vivo validation of [ 89 Zr]Zr-DFO-muAR9.6 and [ 89 Zr]Zr-DFO-huAR9.6 in murine models of MUC16-positive HGSOC and PDAC. Both radioimmunoconjugates displayed excellent tumor-targeting properties in vivo, ultimatelyResearch.

MUC16 is a membrane bound glycoprotein involved in the progression and metastasis of pancreatic and ovarian cancer. The protein is shed into the serum and the resulting cancer antigen 125 (CA125) can be detected by immunoassays. The CA125 epitope is used for monitoring ovarian cancer treatment progression, and has emerged as a potential target for antibody mediated immunotherapy. The extracellular tandem repeat domain of the protein is composed of repeating segments of heavily glycosylated sequence intermixed with homologous SEA (Sperm protein, Enterokinase and Agrin) domains. Here we report the purification and the first X-ray structure of a human MUC16 SEA domain. The structure was solved by molecular replacement using a Rosetta generated structure as a search model. The SEA domain reacted with three different MUC16 therapeutic antibodies, confirming that the CA125 epitope is localized to the SEA domain. The structure revealed a canonical ferredoxin-like fold, and contained a conserved disulfide bond. Analysis of the relative solvent accessibility of side chains within the SEA domain clarified the assignment of N-linked and O-linked glycosylation sites within the domain. A model of the glycosylated SEA domain revealed two major accessible faces, which likely represent the binding sites of CA125 specific antibodies. The results presented here will serve to accelerate future work to understand the functional role of MUC16 SEA domains and antibody recognition of the CA125 epitope.

ObjectiveTo determine the X-ray structure and biophysical properties of a Camelid VHH isolated from a naïve phage display library.ResultsSingle domain antibodies (VHH) derived from the unique immune system of the Camelidae family have gained traction as useful tools for biotechnology as well as a source of potentially novel therapeutics. Here we report the structure and biophysical characterization of a VHH originally isolated from a naïve camelid phage display library. VHH R419 has a melting temperate of 66 °C and was found to be a monomer in solution. The protein crystallized in space group P6522 and the structure was solved by molecular replacement to a resolution of 1.5 Å. The structure revealed a flat paratope with CDR loops that could be classified into existing canonical loop structures. A combination of high expression yield, stability and rapid crystallization might make R419 into a candidate scaffold for CDR grafting and homology modeling.

The integral membrane glycoprotein Mucin‐16 (MUC16) has emerged as an important cancer antigen that displays a high degree of tumor selectivity. MUC16 is often overexpressed and involved in tumorigenesis in several malignancies, including pancreatic and ovarian cancer. The role of MUC16 in cancer progression is complex and provides multiple points for therapeutic intervention. However, despite clinical interest in MUC16 as an immunotherapy target, surprisingly little is known regarding how antibodies bind the protein. Here we report the humanization, epitope mapping, and structure determination of a MUC16 specific therapeutic antibody. The antibody was humanized using a germline complementary determining region (CDR) loop grafting approach and produced by transient transfection in Chinese hamster ovary (CHO) cells. The humanized and murine antibodies displayed nearly identical binding affinity to a recombinant MUC16 SEA (Sperm protein, Enterokinase, and Agrin) domain as measured by enzyme linked‐immunosorbent assay (ELISA) and surface plasmon resonance (SPR). High‐resolution x‐ray structures of the antibodies indicated no significant changes associated with humanization. Initial epitope mapping using an ELISA and overlapping MUC16 constructs suggested that the antibody epitope was localized to a SEA domain and was non‐linear and conformational in nature. A more detailed epitope mapping study carried out using hydrogen‐deuterium exchange mass‐spectrometry revealed five regions on the SEA domain that resulted in reduced deuteration when the antibody was bound to the antigen. These results were confirmed by x‐ray structures of the murine and humanized antibodies complex with the SEA domain. The structures revealed a complex, non‐linear structural epitope with a long b‐hairpin forming the center of the interaction. Finally, a fully glycosylated recombinant SEA domain was produced by transient transfection in CHO cells and used to assess the role of MUC16 glycosylation on antibody binding. Surprisingly, the densely glycosylated SEA domain bound the antibody with approximately 2‐fold higher affinity compared to the unglycosylated domain, suggesting a role for antigen glycosylation in mediating antibody binding. The results presented here represent the first structural characterization of an antibody in complex with MUC16 and reveal a complex, non‐linear epitope influenced by glycosylation. These findings will help to accelerate the clinical development of this promising therapeutic agent.